Berberine bridge enzyme & other flavin-dependent plant oxidases
Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD moiety, which is essential for catalysis. The reaction involves the oxidation of the N-methyl group of the substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation of a carbon-carbon bond (the “berberine bridge”). The ultimate acceptor of the substrate-derived electrons is dioxygen, which reoxidizes the flavin to its resting state:
The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the oxidative cyclization reaction by a biochemical and structural approach.
We have developed a new expression system for BBE (using cDNA from Eschscholzia california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca. 500 mg from a 10-L culture). The availability of suitable quantities of BBE enabled us to crystallize the protein and to solve the structure in collaboration with Prof. Karl Gruber at the Karl-Franzens University Graz (see below).
Based on the three-dimensional structure of BBE, we have performed a site-directed mutagenesis program to investigate the role of amino acids present in the active site of the enzyme. In conjunction with other experiments, this has led to the formulation of a new reaction mechanism for the enzyme (thesis project of Andreas Winkler). Currently, Silvia Wallner (PhD student) studies the functional role of several amino acid residues interacting with the isoalloxazine ring of the flavin cofactor. In addition, Silvia and Corinna Dully (master student) investigate whether alternative covalent modifications in the 8-alpha--position are feasible, i.e. whether aspartate or tyrosine can form a covalent bond to the 8-alpha-methyl group. In collaboration with Prof. Toni Kutchan at the Donald Danforth Plant Science Center in St. Louis, we have identified other plant genes that apparently encode flavin-dependent oxidases.